Cord blood (CB) is a source of hematopoietic stem cells (HSC) for patients with hematological diseases and disorders that do not have an HLA matched bone marrow donor. Two impediments to the successful use of CB transplantation are the low number of HSC in CB and the high rate of infection due to delayed establishment of immunocompetence. It is hypothesized that supplementing CB transplants with ex vivo- expanded lymphoid progenitors will increase transplant cell number and provide a source of rapidly engrafting antigen-responsive cells while CB HSC mature in the bone marrow. Recently, in vitro culture conditions for expanding T cell progenitors from CB HSC on a human non-tumorogenic thymic epithelial cell line (TEC) have been discovered. The first aim of this project is to expand large numbers of B cell progenitors from CB HSC which, in combination with T cell progenitors, should provide complete adaptive immune protection for transplant patients. In addition to adapting TEC cultures to support B cell development, we will explore whether adherent cells of the omentum, a site of fetal B lymphopoiesis, can provide an appropriate microenvironment for the expansion and differentiation of CB HSC. The second aim of this project is to test whether in vitro generated lymphoid progenitors engraft rapidly and are functional. The capacity of the ex vivo-generated lymphoid progenitors to rapidly engraft will be assessed by injecting irradiated, immunodeficient NOD/SCID/IL2Rgamma null mice with lymphoid cells from either cultured CB or uncultured CB. Lymphocyte function in reconstituted mice challenged with a T-dependent and T- independent antigen will be assessed by: 1) measuring antibody production by ELISA and 2) performing CDR3 spectratype analysis to determine whether or not the response to antigen is polyclonal. PUBLIC HEALTH RELEVANCE: Cord blood transplantation is a therapy for immunodeficiency disorders, congenital anemia, leukemia, and other hematopoietic diseases and disorders. The aims of this project support the long-term goal of improving the success of cord blood transplantation by increasing the number of transplanted cells and rapidly establishing immunocompetence in transplant patients. We anticipate that achievement of this goal will reduce mortality due to post-transplant infection and make cord blood transplants available to patients of all ages, ethnicities, and both genders.